PROJECT SUMMARY Atopic dermatitis (AD) is an inflammatory skin disease with compromised innate immunity as indicated by susceptibility to bacterial and viral infection. Despite the identification of genetic polymorphisms in genes in the innate immune signaling pathways, such as TLR1/2/6/9 and NOD1/2, the role of defective innate immunity in AD pathogenesis remains poorly understood. In this study, we provide new evidence to support the contribution of defective innate immunity in Th2 activation and AD pathogenesis with Trim32 knockout mice as a model. In response to topical application of imiquimod, a toll-like receptor agonist used in an established psoriasis model, we observed that Trim32-deficient mice instead developed AD-like phenotypes, including dermal infiltration of eosinophils and mast cells, increased Th2 cytokine production, and elevated serum IgE, compared to the expected Th17 response in Trim32-competent wild type littermates. Based upon this, we hypothesize that Trim32 (an E3 ubiquitin ligase with innate anti-viral activity) regulates homeostasis in the host response to infection and, further, that defects in Trim32 pathway proteins or functionality can predispose to Th2 atopic phenotypes. In seeking Trim32 substrate proteins potentially responsible for atopic features in these mice and in human AD, we provide novel evidence for Trim32 regulation of protein kinase PKC?, a known Stat6 activator of a signaling cascade culminating in IL-4/IL-5 expression in T cells and IgE production by B cells in allergy models in mice. We therefore propose to address the role of Trim32/ PKC? axis in the regulation of molecular signaling events that are responsible for the development of AD-like disease in mouse models, in parallel to validation of findings in human AD patient samples. In view of our discoveries, we propose the following aims: 1) Define the role of Trim32 in regulating a PKC? mediated IL-4/Stat6 signaling pathway in mouse models; 2) Investigate the molecular and structural basis of a homeostatic negative regulatory axis of Trim32 and PKC?; and 3) Evaluate the Trim32 and PKC? axis in association with AD severity and AD susceptibility to viral infection in humans. Our studies will incorporate Multiplex Immunohistochemistry Image Cytometry for analysis of human skin biopsies and advanced imaging technologies to identify localization of interacting proteins by super resolution microscopy, correlative light and electron microscopy, and molecular structure analysis. This study will provide new insight for better understanding of the molecular mechanisms of Th2 polarization underlying AD pathology and promises to improve prediction of patient severity of disease and treatment response and to suggest novel intervention strategies to restore healthy immune and skin homeostasis in AD patients.